Therapeutic uses of humanized antibodies against alpha-4 integrin

Abstract

The invention provides methods of treatment using humanized immunoglobulins that specifically bind to alpha-4 integrin. The methods are useful for treatment of asthma, atherosclerosis, AIDS dementia, diabetes, inflammatory bowel disease, rheumatoid arthritis, transplant rejection, graft versus host disease, tumor metastasis, nephritis, atopic dermatitis, psoriasis, myocardial ischemia, and acute leukocyte mediated lung injury.

1 - 26 . (canceled) 
     
     
         27 . A humanized immunoglobulin comprising a humanized heavy chain and a humanized light chain:
 (1) the humanized light chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the mouse 21-6 immunoglobulin light chain variable domain designated SEQ. ID. NO: 2, and a variable region framework from a human kappa light chain variable region framework sequence except in at least one position selected from a first group consisting of L45, L49, L58 and L69, wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse 21-6 immunoglobulin light chain variable region framework; and   (2) the humanized heavy chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the mouse 21-6 immunoglobulin heavy chain variable domain designated SEQ. ID. NO: 4, and a variable region framework from a human heavy chain variable region framework sequence except in at least one position selected from a second group consisting of H27, H28, H29, H30, H44, H71, wherein the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse 21-6 immunoglobulin heavy chain variable region framework;   wherein the humanized immunoglobulin specifically binds to VLA-4 with a binding affinity having a lower limit of about 10 7  M 4  and an upper limit of about five-times the binding affinity of the mouse 21-6 immunoglobulin.   
     
     
         28 . The humanized immunoglobulin of  claim 27 , wherein the humanized light chain variable region framework is from an RE1 variable region framework sequence except in at least one position selected from the first group, and except in at least one position selected from a third group consisting of positions L104, L105 and L107, wherein the amino acid position is occupied by the same amino acid present in the equivalent position of a kappa light chain from a human immunoglobulin other than RE1. 
     
     
         29 . The humanized immunoglobulin of  claim 28 , wherein the humanized heavy chain variable region framework is from a 21/28′CL variable region framework sequence. 
     
     
         30 . The humanized immunoglobulin of  claim 29 , wherein the humanized light chain variable region framework comprises at least three amino acids from the mouse 21.6 immunoglobulin at positions in the first group and three amino acids from the kappa light chain from the human immunoglobulin other than RE1 at positions in the third group, and the humanized heavy chain variable region framework comprises at least five amino acids from the mouse 21.6 immunoglobulin at positions in the second group. 
     
     
         31 . The humanized immunoglobulin of  claim 30 , wherein the humanized light chain variable region framework is identical to the RE1 light chain variable region framework sequence except for the at least three positions from the first group and the three positions from the third group, and the heavy chain variable region framework is identical to the 21/28′CL heavy chain variable region framework sequence except for the at least five positions from the second group. 
     
     
         32 . The humanized immunoglobulin of  claim 31 , wherein the at least three positions from the first group are positions L45, L58 and L69, and at the least five positions from the second group are positions H27, H28, H29, H30 and H71. 
     
     
         33 . The humanized immunoglobulin of  claim 32 , wherein the humanized light chain comprises complementarity determining regions that are identical to the corresponding complementarity determining regions of the mouse 21-6 heavy chain, and the humanized heavy chain comprises complementarity determining regions that are identical to the corresponding complementarity determining regions of the mouse 21-6 heavy chain, except that the CDR3 region of the humanized heavy chain may or may not comprise a phenylalanine residue at position H98. 
     
     
         34 . The humanized immunoglobulin of  claim 33 , wherein the CDR3 of the humanized heavy chain comprises a phenylalanine residue at position H98. 
     
     
         35 . The humanized immunoglobulin of  claim 27 , wherein the amino acid sequence of the mature light chain variable region is selected from the sequence designated La (SEQ. ID. NO:7) in  FIG. 6  or the sequence designated Lb (SEQ. ID. NO:8) in  FIG. 6 . 
     
     
         36 . The humanized immunoglobulin of  claim 27 , wherein the amino acid sequence of the mature heavy chain variable region is selected from the sequence designated Ha (SEQ. ID. NO:11) in  FIG. 7 , the sequence designated Hb (SEQ. ID. NO: 12) in  FIG. 7 , or the sequence designated Hc (SEQ. ID. NO:13) in  FIG. 7 . 
     
     
         37 . The humanized immunoglobulin of  claim 35 , wherein the amino acid sequence of the mature light chain variable region is the sequence designated La (SEQ. ID. NO: 7) and the mature heavy chain variable region is selected from Ha (SEQ. ID. NO:11) or Hc (SEQ. ID. NO: 13). 
     
     
         38 . An antigen-specific binding fragment of the humanized immunoglobulin of  claim 37 . 
     
     
         39 . A humanized immunoglobulin of  claim 37 , further comprising a constant region domain. 
     
     
         40 . A humanized immunoglobulin of  claim 39 , wherein the constant region domain has an effector function. 
     
     
         41 . The humanized immunoglobulin of  claim 37 , wherein the humanized immunoglobulin is a Fab fragment. 
     
     
         42 . A nucleic acid encoding a heavy chain of a humanized antibody of claim  1  or an antigen-specific binding fragment thereof, or a light chain of a humanized antibody of  claim 27 , or an antigen-specific binding fragment thereof. 
     
     
         43 . A pharmaceutical composition comprising a humanized antibody of  claim 37 , or a binding fragment thereof, and a pharmaceutically acceptable carrier therefor. 
     
     
         44 . A method for detecting VLA-4 antigen, the method comprising:
 administering a humanized immunoglobulin of  claim 37 , or an antigen-specific binding fragment thereof, to a tissue sample from a patient; and   detecting complexes formed by specific binding between the antibody or fragment and VLA-4 present in the target sample.   
     
     
         45 . A method of inhibiting adhesion of a leukocyte to an endothelial cell, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of  claim 43 . 
     
     
         46 . A method of treating a disease in a patient comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of  claim 43 , wherein the disease is selected from multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, rheumatoid arthritis, asthma, diabetes, atherosclerosis, AIDS dementia, transplant rejection, graft versus host disease, tumor metastasis, nephritis, atopic dermatitis, psoriasis, myocardial ischemia, and acute leukocyte mediated lung injury.